The science of genetics is based on the identification and characterization of mutations, which are changes in DNA (DNA polymorphisms) due to nucleotide substitution, insertion, or deletion. Many techniques have been developed to compare homologous segments of DNA to determine if the segments are identical or if they differ at one or more nucleotides. Practical applications of these techniques include genetic disease diagnoses, forensic techniques, human genome mapping and agricultural applications.
The most definitive method for comparing DNA segments is to determine the complete nucleotide sequence of each segment. Examples of how sequencing has been used to study mutations in human genes are included in the publications of Engelke et al., Proc. Natl. Acad. Sci. U.S.A., 85:544-548 (1988) and Wong et-al., Nature 330:384-386 (1987). At the present time, it is not practical to use extensive sequencing to compare more than just a few DNA segments, because the effort required to determine, interpret, and compare sequence information is extensive.
For genetic mapping purposes, the most commonly used screen for DNA polymorphisms arising from mutation consists of digesting DNA with restriction endonucleases and analyzing the resulting fragments, as described by Botstein et al., Am. J. Hum. Genet., 32:314-331 (1980); White, et al., Sci Am., 258:40-48 (1988). Mutations that affect the recognition sequence of the endonuclease will preclude enzymatic cleavage at that site, thereby altering the cleavage pattern of the DNA. DNAs are compared by looking for differences in restriction fragment lengths. A major drawback to this method (known as restriction fragment length polymorphism mapping or RFLP mapping) is its inability to detect mutations that do not affect cleavage with a restriction endonuclease. Thus, many mutations are missed with this method. One study by Jeffreys, Cell, 18:1-18 (1979), was able to detect only 0.7% of the mutational variants estimated to be present in a 40,000 base pair region of human DNA. Another difficulty is that the methods used to detect restriction fragment length polymorphisms are very labor intensive, in particular, the techniques involved with Southern blot analysis.
The primer extension process described in Proudfoot et al., Science 209:1329-1336 (1980), has been widely used to study the structure of RNA and also has been used to characterize DNA, see, e.g., Engelke et al., Proc. Natl. Acad. Sci. U.S.A., 85:544-548 (1988). This process consists of hybridizing a labeled oligonucleotide primer to a template RNA or DNA and then using a DNA polymerase and deoxynucleoside triphosphates to extend the primer to the 5' end of the template. The labeled primer extension product is then fractionated on the basis of size, usually by electrophoresis through a denaturing polyacrylamide gel. When used to compare homologous DNA segments, this process can detect differences due to nucleotide insertion or deletion. Because size is the sole criterion used to characterize the primer extension product, this method cannot detect differences due to nucleotide substitution.
Mullis et al., U.S. Pat. No. 4,683,195, and Mullis, U.S. Pat. No. 4,683,202 disclose polymerase chain reactions which can be used to amplify any specific segment of a nucleic acid. These analytical methods have been used to detect polymorphisms through amplification of selected target DNA segments from test genomes. A drawback to such methods is the requirement that a sufficient number of bases at both ends of the specific segment be known in sufficient detail so that two oligonucleotide primers can be designed which will hybridize to different strands of the target segment. It is labor intensive to obtain the necessary sequence information from target genomes in order to design the necessary primers. M. H. Skolnic and R. B. Wallace, Simultaneous Analysis of Multiple Polymorphic Loci Using Amplified Sequence Polymorphisms (ASPs), Genomics 2:273-278.
Amplification using oligonucleotides of random sequence is described in a theoretical approach for mapping a genome of a higher eukaryote in "Happy Mapping: a Proposal for Linkage Mapping the Human Genome," P. H. Dear and P. R. Cook, Nucleic Acids Research, Vol. 17, No. 17, 6795-6807 (1989). The authors propose to identify the relationship between 5,000 different DNA segments amplified from human genomic DNA by performing a polymerase chain reaction (PCR) with various pairwise combinations of arbitrary-sequence primers. The template DNA is isolated separately from single haploid cells of an individual organism. Their method suggests generating a map describing the physical location of DNA regions giving rise to the amplified products in an individual organism. This would be accomplished by identifying PCR products which co-amplify from the same piece of DNA, thus establishing their physical proximity. In Dear and Cook's method, polymorphisms would be excluded from the analysis. Their idea requires the identification of the same DNA segment in many different haploid cells, and polymorphisms defeat this requirement. Although efficient at describing the physical relationship between any two PCR products, happy mapping cannot describe the location of any genetic traits of interest. This can only be accomplished by correlating the segregation of a genetic trait with many different polymorphisms through a sexual cross between two individuals. The authors teach that primer pairs longer than 9-10 nucleotides are necessary as primer pairs of 9-10 nucleotides would prime inefficiently.
The present invention provides a process for the detection of genetic polymorphisms in random nonspecific nucleic acid segments. The process utilizes a reaction primed by an oligonucleotide(s) conveniently prepared with no knowledge of the base sequence of segments amplified.